Transendothelial migration (TEM) is a critical step in the inflammatory response in which leukocytes leave the blood vessel to enter the inflamed tissues. Most pathology, including ischemia/reperfusion (I/R) injury, is due to dysfunctional inflammation. I/R injury occurs when tissues deprived of oxygen are reoxygenated, producing reactive oxygen species that damage cells and incite local inflammation. I/R injury is a major source of morbidity and mortality in myocardial infarction (MI; heart attack), making ischemic injury to the heart muscle even worse. Limiting I/R injury is major goal in treatment of MI and other vascular disorders. TEM is initiated by interactions between platelet/endothelial cell adhesion molecule 1 (PECAM) on the surface of leukocytes and PECAM concentrated at the endothelial cell (EC) borders. Efficient TEM requires a transient increase in cytosolic free calcium ion concentration (?[Ca2+]i). TRPC6 was found to be the calcium channel responsible for (?[Ca2+]i) and functions downstream of PECAM-PECAM interactions to promote TEM. However, the mechanism by which PECAM signals to TRPC6 is unknown. I will determine how PECAM signals to TRPC6, how TRPC6 regulates ?[Ca2+]i in vivo, and the relevance of TRPC6 to I/R injury in MI. Endothelial cells have a junctional mechanosensory complex consisting of PECAM, vascular endothelial (VE)-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). This signaling complex responds to fluid shear. Preliminary data suggest that this same complex may also function to transmit signals from PECAM to TRPC6 during TEM. Aim I will determine how PECAM signals to activate TRPC6. We will use specific pharmacologic inhibitors as well as knockdown and re-expression strategies for each component of the system. PECAM FRET tension sensors and VE-cadherin mutants will be used to test the hypothesis that EC PECAM uses the same mechanosensory signaling complex to transmit stress from engagement of PECAM on the leukocyte pseudopod as it does to transmit stress from fluid sheer. In Aim II, intravital microscopy will be used to quantify the timing and intensity of ?[Ca2+]i in mice with the genetically encoded calcium sensor GCaMP3 expressed in EC. We will breed TRPC6-deficient and sufficient mice with GCaMP3 restricted to EC. These mice will be irradiated and reconstituted with bone marrow from TRPC6 WT mice to compare TEM and ?[Ca2+]i in mice with and without TRPC6. Aim III will determine the role of TRPC6 in the clinically relevant inflammatory setting of ischemia/reperfusion (I/R) injury. We will study I/R at the cellular level by intravital microscopy and at the organ level in an acute myocardial infarction model. As TRPC6 knockout significantly decreases TEM in models of inflammation, we hypothesize that TRPC6 inhibition will offer protection from I/R injury. These studies will provide insights into the mechanisms of TEM and may identify TRPC6 as a novel therapeutic target for a multitude of diseases caused by pathologic inflammation, including I/R injury in myocardial infarction.